Secretion is one of the most ubiquitous of cellular processes. Regulated exocytosis is central to such diverse biological functions as information processing, reproduction, motility, temperature regulation, metabolism, the immune response, and signal transduction, but the elucidation of its mechanism(s) remains a challenge to cell physiologists. The neurohypophysis is a classic in vitro preparation, uniquely relevant for studying evoked release of peptide hormones. Magnocellular neurons from the supraoptic and paraventricular nuclei in the hypothalamus project their axons as bundles of fibers through the median eminence and infundibular stalk to arborize extensively and terminate in the neurohypophysis, a quasi-pure collection of nerve terminals, where the neuropeptides oxytocin and vasopressin are released into the circulation. Despite remarkable progress in identifying components of the macromolecular machinery essential for their release, direct observation in real time of the intraterminal events that follow electrical excitation, but precede membrane fusion, has proven extremely difficult. To identify these steps we have developed an arsenal of biophysical techniques possessing time scales ranging from microseconds to minutes, with emphasis on the former. Three of them, in particular, constitute the core of each of our specific aims: 1) High Bandwidth Dynamic Atomic Force Microscopy (HBDAFM), to study the rapid, but very small (~1.0 ), mechanical events (spike and dip) which accompany neuropeptide secretion in mammalian nerve terminals. 2) Real-time detection of Ca2+-transients, from whole terminals as well as from near- membrane regions, to identify intermediate steps between Ca2+-entry and exocytosis. 3) Millisecond time resolved optical detection of secretion per se. These technologies, already at different stages of development in our laboratory, will be combined in this proposal to capture, in a single experiment, sequential, albeit partially overlapping events, which are essential components of excitation-secretion coupling. PUBLIC HEALTH RELEVANCE: The hormones secreted by the neurohypophysis, oxytocin and vasopressin, are implicated in functions as diverse as blood pressure regulation, water balance, milk ejection, social cognition, sexual behavior, and mood control. Therefore, greater insight into arginine vasopressin exocytosis, for example, may enhance our ability to treat hypertension~ furthermore, since many neurological ailments involve an impairment of neurotransmitter release, advancing our understanding of its presynaptic component is key to the development of new approaches to the treatment of disorders of synaptic transmission implicated in ailments as devastating as, to name a few, Lambert-Eaton Syndrome, schizophrenia, Alzheimer's disease and depression.